Tinostamustine for use in the treatment of t-cell prolymphocytic leukaemia

ABSTRACT

T-PLL Therapy There is provided tinostamustine or a pharmaceutically acceptable salt thereof for use in the treatment of T-cell prolymphocytic leukemia (T-PLL) in a patient in need thereof.

TECHNICAL FIELD

The present invention relates to a method of treating T-cellprolymphocytic leukemia (T-PLL).

BACKGROUND TO THE INVENTION

Cancer is one of the most life threatening diseases. Cancer is acondition in which cells in a part of the body experience out-of-controlgrowth. According to latest data from American Cancer Society, it isestimated there will be 1.69 million new cases of cancer in USA in 2017.Cancer is the second leading cause of death in the United States (secondonly to heart disease) and will claim more than 601,000 lives in 2017.In fact, it is estimated the average lifetime risk of developing canceris 40.8% for American males and 37.5% for American women. Thereforecancer constitutes a major public health burden and represents asignificant cost in the United States. These figures are reflectedelsewhere across most countries globally, although the types of cancerand relative proportions of the population developing the cancers varydepending upon many different factors such including genetics and diet.

For decades surgery, chemotherapy, and radiation were the establishedtreatments for various cancers. Patients usually receive a combinationof these treatments depending upon the type and extent of their disease.But chemotherapy is the most important option for cancer patients whensurgical treatment (i.e. the removal of diseased tissue) is impossible.While surgery is sometimes effective in removing tumours located atcertain sites, for example, in the breast, colon, and skin, it cannot beused in the treatment of tumours located in other areas, such as thebackbone, nor in the treatment of disseminated hematologic cancersinclude cancers of the blood and blood-forming tissues (such as the bonemarrow). They include multiple myeloma, lymphoma and leukemia. Radiationtherapy involves the exposure of living tissue to ionizing radiationcausing death or damage to the exposed cells. Side effects fromradiation therapy may be acute and temporary, while others may beirreversible. Chemotherapy involves the disruption of cell replicationor cell metabolism. It is used most often in the treatment of breast,lung, and testicular cancer. One of the main causes of failure in thistreatment of cancer is the development of drug resistance by the cancercells, a serious problem that may lead to recurrence of disease or evendeath. Thus, more effective cancer treatments are needed.

Leukemia is a cancer of the blood cells. Leukemias begin in theblood-forming tissue of the bone marrow. The cancers do not form solidtumours but instead large numbers of abnormal white blood cells(leukemia cells and leukemic blast cells) build up in the blood and bonemarrow. There are four main types of leukemia: Acute myeloid leukemia(AML), Chronic myeloid leukemia (CML), Acute lymphocytic leukemia (ALL),and Chronic lymphocytic leukemia (CLL).

T-cell prolymphocytic leukemia (T-PLL) is recognised in the WHOclassification of hematologic malignancies as a leukemic peripheralT-cell neoplasm and is of mature T-cell phenotype. Although representingthe most frequent mature T-cell leukemia, T-PLL is nevertheless anextremely uncommon hematological malignancy and is rarely encountered indaily routine (with incidence of ˜0.6/million). T-PLL also has a verypoor prognosis, with the median overall survival of patients with T-PLLbeing around 7 months with conventional chemotherapy.

Patients with T-PLL typically present with exponentially risinglymphocyte counts in peripheral blood accompanied by lymphadenopathywith hepatosplenomegaly, and bone marrow involvement.

T-PLL characteristically shows rapid progression and does not respondwell to standard multi-agent chemotherapy. The monoclonal anti-CD52antibody alemtuzumab was the only (targeted) agent that was shown toinduce a high rate of remission, albeit with relapse the rule.Alemtuzumab had overall response rates ranging from 51-95% with a mediansurvival of 15-19 months in patients achieving a complete response.

However, alemtuzumab was withdrawn from the market in 2012 and there iscurrently no effective first line treatment for T-PLL.

There is therefore a need for effective chemotherapeutic treatments ofT-PLL.

In WO-A-2010/085377, the compound of formula I below is disclosed. It isa first-in-class dual-functional alkylating-HDACi fusion molecule whichpotently inhibits the HDAC pathway.

Biological assays showed that the compound of formula I potentlyinhibits HDAC enzyme (HDAC1 IC₅₀ of 9 nM). The compound of formula I hasan INN of tinostamustine and is also known in the art as EDO-S101. It isan AK-DAC (a first-in-class alkylating deacetylase molecule) that, inpreclinical studies, has been shown to simultaneously improve access tothe DNA strands within cancer cells, break them and block damage repair.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is providedtinostamustine or a pharmaceutically acceptable salt thereof for use inthe treatment of T-cell prolymphocytic leukemia (T-PLL).

It has surprisingly been discovered that tinostamustine or apharmaceutically acceptable salt thereof is particularly effective inthe treatment of T-PLL, with activity data showing strong in vitro andin vivo sensitivity to this compound. Thus, the need for a new andeffective treatment of T-PLL is met by the present invention.

In a further aspect of the present invention there is provided use oftinostamustine or a pharmaceutically acceptable salt thereof for themanufacture of a medicament for the treatment of T-PLL.

In a further aspect of the present invention there is provided a methodof treating T-PLL in a patient in need thereof comprising administeringto said patient an effective amount of tinostamustine or apharmaceutically acceptable salt thereof.

In a further aspect of the present invention there is provided a kitcomprising tinostamustine or a pharmaceutically acceptable salt thereoftogether with instructions for treating T-PLL.

The following features apply to all aspects of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a plot of HH cell viability relative to control afterexposure to increasing concentrations of the HDAC inhibitor SAHA(vorinostat), bendamustine, SAHA+bendamustine, and EDO-S101;

FIG. 1b shows a dose response curve for the SAHA (vorinostat),bendamustine, SAHA+bendamustine, and EDO-S101, in HH cells.

FIG. 2 shows western blots analysis of patient T-PLL samples showing theeffect of SAHA (vorinostat), bendamustine, SAHA+bendamustine, andEDO-S101 on various markers relevant to T-PLL.

FIG. 3a is a dose response curve showing the relative number of livingT-PLL cells in suspension culture after 48 h treatment comparing SAHA(vorinostat), bendamustine, SAHA+bendamustine, and EDO-S101;

FIGS. 3b and 3c show the effect of increasing concentrations of SAHA(vorinostat), bendamustine, SAHA+bendamustine, and EDO-S101, on primaryT-PLL cells with (FIG. 3c ) and without (FIG. 3b ) co-cultures of thehuman bone marrow stromal cell line NKtert.

FIG. 3d shows the effect of increasing concentrations of SAHA(vorinostat), bendamustine, SAHA+bendamustine, and EDO-S101, on NKtertcell viability.

FIGS. 4a 4b and 4c show the results of a transfer model for CD2-MTCP1p13 mice investigating fludarabine, bendamustine and EDO-S101.

FIG. 5 shows the results of a transfer model for ΔJAK1 miceinvestigating fludarabine, bendamustine and EDO-S101.

DETAILED DESCRIPTION OF THE INVENTION

In the present application, a number of general terms and phrases areused, which should be interpreted as follows.

The compound of formula I has an INN of tinostamustine and is also knownin the art as EDO-S101. The IUPAC name is7-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-hydroxyheptanamide.

“Patient” includes humans, non-human mammals (e.g., dogs, cats, rabbits,cattle, horses, sheep, goats, swine, deer, and the like) and non-mammals(e.g., birds, and the like).

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids, or withorganic acids. Pharmaceutically acceptable salts also include baseaddition salts which may be formed when acidic protons present arecapable of reacting with inorganic or organic bases. Generally, suchsalts are, for example, prepared by reacting the free acid or base formsof these compounds with a stoichiometric amount of the appropriate baseor acid in water or in an organic solvent or in a mixture of the two.Generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol or acetonitrile are preferred. Examples of the acid additionsalts include mineral acid addition salts such as, for example,hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, and organic acid addition salts such as, forexample, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate,succinate, tartrate, salicylate, tosylate, lactate,naphthalenesulphonae, malate, mandelate, methanesulfonate andp-toluenesulfonate. Examples of the alkali addition salts includeinorganic salts such as, for example, sodium, potassium, calcium andammonium salts, and organic alkali salts such as, for example,ethylenediamine, ethanolamine, N,N-dialkylenethanolamine,triethanolamine and basic aminoacids salts.

In the present invention, the pharmaceutically acceptable salt oftinostamustine may preferably be the hydrochloride, hydrobromide,hydroiodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate,malate, maleate, oxalate, succinate, fumarate, tartrate, tosylate,mandelate, salicylate, lactate, p-toluenesulfonate, naphthalenesulfonateor acetate salt.

It has been found that tinostamustine or a pharmaceutically acceptablesalt thereof shows surprising efficacy in T-PLL. In particular, it hasbeen found that tinostamustine or a pharmaceutically acceptable saltthereof is useful in the treatment of T-PLL.

T-cell prolymphocytic leukemia or T-PLL is a leukemic peripheral T-cellneoplasm and is of mature T-cell phenotype (Campo, E et al, Blood 117,2011 5019-32). Although representing the most frequent mature T-cellleukemia, T-PLL is nevertheless an extremely uncommon hematologicalmalignancy and is rarely encountered in daily routine (with incidence of˜0.6/million). T-PLL also has a very poor prognosis, with the medianoverall survival of patients with T-PLL being around 7 months withconventional chemotherapy.

Patients with T-PLL typically present with exponentially risinglymphocyte counts in peripheral blood accompanied by lymphadenopathywith hepatosplenomegaly, and bone marrow involvement.

The therapeutically effective amount of tinostamustine or apharmaceutically acceptable salt administered to the patient is anamount which confers a therapeutic effect in accordance with the presentinvention on the treated subject, at a reasonable benefit/risk ratioapplicable to any medical treatment. The therapeutic effect may beobjective (i.e. measurable by some test or marker) or subjective (i.e.subject gives an indication of or feels an effect). An effective amountof tinostamustine or a pharmaceutically acceptable salt thereofaccording to the present invention is believed to be one whereintinostamustine or a pharmaceutically acceptable salt thereof is includedat a dosage range of from 0.3 mg/m² to 300 mg/m² body surface area ofthe patient or from 20 mg/m² to 150 mg/m² body surface area of thepatient.

The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the severity ofthe disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination orcontemporaneously with the specific compound employed; and like factorswell known in the medical arts.

“Metastatic Cancer”. Cancer has the ability to spread within the body.Cancer cells can spread locally by moving into nearby normal tissue.Cancer can also spread regionally, to nearby lymph nodes, tissues, ororgans. Cancer can therefore spread to distant parts of the body. Whenthis happens, it is called metastatic cancer (also known as stage IVcancer), and the process by which cancer cells spread to other parts ofthe body is called metastasis. Thus, in metastasis, cancer cells breakaway from where they first formed (primary cancer), travel through theblood or lymph system, and form new tumours (metastatic tumours) inother parts of the body.

Metastatic cancer cells have features like that of the primary cancerand not like the cells in the place where the cancer is found. Thisenables doctors to tell whether a cancer is metastatic. Metastaticcancers are given the same name as the primary cancer. For example,breast cancer that has spread to the lung is called metastatic breastcancer, not lung cancer. It is treated as stage IV breast cancer, not aslung cancer.

Metastatic T-PLL refers to a T-cell prolymphocytic leukemia that hasmetastasised to a new location in the body. The cancer is treated as astage IV T-PLL cancer.

“Advanced Cancer” is a cancer that is not curable but responds totreatment. Disease directed therapy is still very important because itprolongs life. For terminal cancer, therapy cannot prolong survivalsignificantly due to the progressive nature of the disease andpalliative care is the main treatment option.

Suitable examples of the administration form of tinostamustine or apharmaceutically acceptable salt thereof include without limitationoral, topical, parenteral, sublingual, rectal, vaginal, ocular, andintranasal. Parenteral administration includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. Preferably, tinostamustine or a pharmaceutically acceptablesalt thereof is administered parenterally, and most preferablyintravenously.

Preferably, tinostamustine or a pharmaceutically acceptable salt thereofis administered intravenously to the patient in need thereof at a dosagelevel to the patient in need thereof of from 0.3 mg/m² to 300 mg/m² bodysurface area of the patient.

Preferably, tinostamustine or a pharmaceutically acceptable salt thereofis administered intravenously to the patient in need thereof at a dosagelevel to the patient in need thereof of from 20 mg/m² to 150 mg/m² bodysurface area of the patient.

It has been found that in embodiments of the present invention,tinostamustine or a pharmaceutically acceptable salt thereof ormedicament comprising the same may preferably be administered to apatient in need thereof on day 1 of each treatment cycle.

Tinostamustine or a pharmaceutically acceptable salt thereof may beadministered on day 1 of a 21 day treatment cycle.

In embodiments according to the present invention, tinostamustine or apharmaceutically acceptable salt thereof or medicament comprising thesame is administered to a patient in need thereof over an infusion timeof 60 minutes; or an infusion time of 45 minutes; or an infusion time of30 minutes.

In embodiments according to the present invention, tinostamustine or apharmaceutically acceptable salt is administered to the patient in needthereof at a dosage level of from 20 mg/m² to 150 mg/m² body surfacearea of the patient, on day 1 of a 21 day treatment cycle, over aninfusion time of 60 minutes.

In embodiments of the present invention, there is provided a kitcomprising tinostamustine or a pharmaceutically acceptable salt thereoftogether with instructions for treating T-PLL.

The instructions may advise administering tinostamustine or apharmaceutically acceptable salt thereof according to variables such asthe state of the T-PLL being treated; the age, body weight, generalhealth, sex and diet of the patient; the time of administration, routeof administration, and rate of excretion of the specific compoundsemployed; the duration of the treatment; drugs used in combination orcontemporaneously with the specific compounds employed; and like factorswell known in the medical arts.

In a further embodiment of the present invention, the patient in need ofsaid treatment is given radiotherapy with (including prior to, during orafter) treatment of the T-PLL with tinostamustine or a pharmaceuticallyacceptable salt thereof. In embodiments of the present invention, thepatient is treated with tinostamustine or a pharmaceutically acceptablesalt thereof and radiotherapy. Preferably, the patient is givenradiotherapy treatment prior to the treatment with tinostamustine or apharmaceutically acceptable salt thereof. The radiotherapy may be givenat a dose of 1 to 5 Gy over 5-10 consecutive days and preferably 2 Gyover 5-10 consecutive days.

In a further embodiment of the present invention, the patient in need ofsaid treatment is given radiotherapy prior to or after treatment of theT-PLL with tinostamustine or a pharmaceutically acceptable salt thereof.Preferably, the patient is given radiotherapy treatment prior to thetreatment with tinostamustine or a pharmaceutically acceptable saltthereof. The radiotherapy may be given at a dose of 1 to 5 Gy over 5-10consecutive days and preferably 2 Gy over 5-10 consecutive days.

When intended for oral administration, tinostamustine or apharmaceutically acceptable salt thereof or medicament comprising thesame may be in solid or liquid form, where semi-solid, semi-liquid,suspension and gel forms are included within the forms considered hereinas either solid or liquid.

Tinostamustine or a pharmaceutically acceptable salt thereof ormedicament comprising the same can be prepared for administration usingmethodology well known in the pharmaceutical art. Examples of suitablepharmaceutical formulations and carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

As a solid composition for oral administration, tinostamustine or apharmaceutically acceptable salt thereof can be formulated into apowder, granule, compressed tablet, pill, capsule, chewing gum, wafer orthe like form. Such a solid composition typically contains one or moreinert diluents or carriers. Any inert excipient that is commonly used asa carrier or diluent may be used in compositions of the presentinvention, such as sugars, polyalcohols, soluble polymers, salts andlipids. Sugars and polyalcohols which may be employed include, withoutlimitation, lactose, sucrose, mannitol, and sorbitol. Illustrative ofthe soluble polymers which may be employed are polyoxyethylene,poloxamers, polyvinylpyrrolidone, and dextran. Useful salts include,without limitation, sodium chloride, magnesium chloride, and calciumchloride. Lipids which may be employed include, without limitation,fatty acids, glycerol fatty acid esters, glycolipids, and phospholipids.

In addition, one or more of the following can be present: binders suchas carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose,or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, corn starchand the like; lubricants such as magnesium stearate; glidants such ascolloidal silicon dioxide; sweetening agents such as sucrose orsaccharin; a flavoring agent such as peppermint, methyl salicylate ororange flavoring; and a coloring agent.

When tinostamustine or a pharmaceutically acceptable salt thereofcompositions is in the form of a capsule (e.g. a gelatin capsule), itcan contain, in addition to materials of the above type, a liquidcarrier such as polyethylene glycol, cyclodextrin or a fatty oil.

Tinostamustine or a pharmaceutically acceptable salt thereofcompositions can be in the form of a liquid, e.g. an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, tinostamustine or a pharmaceutically acceptable saltthereof compositions can comprise one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In tinostamustine or apharmaceutically acceptable salt thereof compositions for administrationby injection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The preferred route of administration is parenteral administrationincluding, but not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,intranasal, intracerebral, intraventricular, intrathecal, intravaginalor transdermal. The preferred mode of administration is left to thediscretion of the practitioner, and will depend in part upon the site ofthe medical condition (such as the site of cancer). In a more preferredembodiment, tinostamustine or a pharmaceutically acceptable salt thereofor medicament comprising the same is administered intravenously.

Liquid forms of tinostamustine or a pharmaceutically acceptable saltthereof or medicament comprising the same, may be solutions, suspensionsor other like form, and can also include one or more of the following:sterile diluents such as water for injection, saline solution,preferably physiological saline, Ringer's solution, isotonic sodiumchloride, fixed oils such as synthetic mono or digylcerides,polyethylene glycols, glycerin, or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; and agents for the adjustmentof tonicity such as sodium chloride or dextrose. A parenteralcombination or composition can be enclosed in an ampoule, a disposablesyringe or a multiple-dose vial made of glass, plastic or othermaterial. Physiological saline is a preferred adjuvant.

Tinostamustine or a pharmaceutically acceptable salt thereof ormedicament comprising the same can be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings, and preferably by bolus.

Examples of compositions comprising tinostamustine or a pharmaceuticallyacceptable salt thereof are disclosed in WO2013/040286.

The present invention may be further understood by consideration of thefollowing non-limiting examples.

EXAMPLES

In the following examples, the compound having the following formula Iis referred to as EDO-S101.

EDO-S101 may be prepared as described in Example 6 of WO-A-2010/085377.

Materials and Methods

EDO-S101 and Control Compounds

EDO-S101 was provided by EDO MundiPharma, and synthesised as describedin Example 6 of WO-A-2010/085377.

Bendamustine was provided by EDO MundiPharma.

Vorinostat (SAHA) (catalogue reference number SML0061-5 mg) andFludarabine were purchased from Sigma-Aldrich.

Cell Culture

RPMI-1640 medium (Sigma-Aldrich) supplemented with 1% L-Glutamine (200mM; Sigma-Aldrich), 10% fetal bovine serum (FBS) (Sigma-Aldrich) andPenicillin/Streptomycin (100 U/0.1M; PAA) was used for in-vitroexperimentation on suspension cultures of primary T-PLL cells, healthyCD3+ T cells, HH cells, and in co-culture experiments with NKtert cells.Cell suspensions were maintained at a density of 1.0×10⁶ cells/mL(primary T-PLL cells) and 2.5×10⁵ cells/mL (HH cells) for all cellculture experiments.

Cells were cultured in a HERAcell incubator (Thermo Scientific Heraeus)at 37° C. and 5% CO₂ with 90% humidity. CD4+ mature T-cell leukemia HHcells were originally isolated from a patient with Sézary Syndrome(Starkebaum et al., 1991). NKtert (human bone marrow stromal cells[BMSC]) were purchased from RIKEN Cell Bank in 2011. Only cells derivedfrom the original cell stock as purchased, and which had been propagatedfor 2 to 3 passages before long-term storage in liquid nitrogen, wereused. Cell cultures were terminated after the 10^(th) passage (4 to 6weeks of being in culture). Cells were authenticated following thawingby evaluation of characteristic growth behaviour and by flow cytometry.Cells were routinely tested for the presence of mycoplasma, usingstandard PCR protocols (primers: for 1: 5′-acaccatgggagytggtaat-3′, (SEQID No; 1) rev1: 5′-cttcwtcgattycagacccaaggcat-3 (SEQ ID NO: 2)′, for 2:5′-gtgsggmtggatcacctcct-3 (SEQ ID NO: 3)′, rev2:5′-gcatccaccawawacyctt-3′(SEQ ID NO: 4)).

Healthy CD3+ T-cells were isolated from healthy human donors.

For co-culture experiments human bone marrow stromal cells (BMSC) NKtertcells (RIKEN BRC, Japan) were seeded at concentrations of 1.5×10⁴cells/well (96 well plate) and incubated at 37° C. in 5% CO₂. After 24hours, NKtert cells at approximately 60-80% confluency were treated with0.02 mg/mL Mitomycin C for 3 hours in RPMI-1460, and then washed twicewith PBS (Life Technologies). After another 24 hours, 4×10⁵ T-PLL cellswere added per well (with and without feeder cell support) and treatedfor 48 hours with the indicated compounds.

In Vitro Drug Treatment and Cell Viability

EDO-S101 (EDO MundiPharma), and vorinostat (SAHA; SML0061-5 mg,Sigma-Aldrich) were dissolved in DMSO. The alkylating agent bendamustine(MundiPharma) was dissolved in methanol. Cells were treated with eachcompound (or compounds) at the indicated concentrations and times.Dosing was based on published ranges and IC₅₀/LD₅₀ titrations. Cellapoptosis was determined using dual staining for Annexin-V (AnxV) and7AAD via flow cytometry.

Human primary T-PLL cells are unsuitable for cultivation under standardlaboratory cell culture conditions, in part, due to their high levels ofgenomic heterogeneity and variable phenotypes. HH cells are derived froma highly chemotherapy resistant cutaneous lymphoma, and are suitable forcultivation under laboratory conditions. HH cells exhibit a comparablephenotype to T-PLL cells, and are therefore frequently used as asurrogate cell line for T-PLL cells for in vitro experiments. HH cellswere therefore selected for the in vitro validation of EDO-S101.

Murine Models

DBA2xC57B6JF1 mice were used as recipients in all experiments.Transplantable leukaemia/lymphoma cells derived from CD2-MTCP1p13 tgmice (Gritti et al, Blood 1998, 92, 268-73; blood, spleen, and bonemarrow) were intraperitoneally injected into background-matched mice (tofacilitate the generation of uniform cohorts). 1×10⁷ cells fromCD2-MTCP1p13 mice were intraperitoneally injected into syngeneicrecipients (n=26). Starting on day 10 post-transplantation, mice with ahomogeneous distribution of leukemic blood leukocytes (WBC) wereselected and randomly assigned into four treatment groups. Each groupwas then treated with either vehicle control (DMSO), fludarabine (34mg/kg days 10, 15, 17, 21), bendamustine (day 10 at 60 mg/kg, days 15,17, 21 at 20 mg/kg), and EDO-S101 (day 10 at 50 mg/kg, days 15, 17, 21at 20 mg/kg) on the indicated days at the indicated doses.

Transplantable leukaemia/lymphoma cells derived from ΔJAK1 mice(Heinrich et al, Mol. Ther. 2013, 21, 1160-8; nodal/spleen mature T-celllymphoma based on insertional mutagenesis activating JAK1) wereintravenously injected into background-matched mice (to facilitate thegeneration of uniform cohorts). 2.5×10⁶ cells were transplantedintravenously into Rag1-deficient mice. Recipients of comparableleukocyte counts were then randomly divided into four treatment groups.Each treatment group was then treated with 18 mg/kg of eitherbendamustine, fludarabine, EDO-S101, or with vehicle control on days 7,10, 13, 17, 22 (DMSO).

Patient Samples

T-PLL cells were isolated from peripheral blood (PB) of T-PLL patientsdiagnosed according to WHO criteria (Swerdlow, S. H. et al Blood 2016,127, 2375-90; Herling et al Blood 2004, 104, 328-335). Diagnosis wasbased on clinical features, immunophenotyping (flow-cytometry andhistochemistry; including TCL1A/MTCP1 expression), FISH/karyotypes, andmolecular studies (TCRmonoclonality). Human tumour samples were obtainedunder institutional review board (IRB)-approved protocols followingwritten informed consent according to the Declaration of Helsinki.Collection and use was approved for research purposes by the ethicscommittee of the University Hospital of Cologne (#11-319). The patientcohort was selected based on uniform front-line treatment (87% of cases)with either single-agent alemtuzumab orfludarabine-mitoxantrone-cyclophosphamide (FMC) plus alemtuzumabchemoimmunotherapy as part of the TPLL120 (NCT00278213) and TPLL2(NCT01186640, unpublished) prospective clinical trials or as included inthe nation-wide T-PLL registry (IRB #12-146) of the German CLL StudyGroup. At diagnosis, patients had a median age of 62 years and included1.5-times more men than women. FISH analysis used standard protocols(Vysis, Abbott).

Western Blot Analysis

T-PLL cells were isolated from peripheral blood (PB) of T-PLL patients.T-PLL cells were cultured in suspension, and treated with eitherbendamustine (1 μM), vorinostat (1 μM), EDO-S101 (1 μM) or an equimolarcombination of vorinostat/bendamustine (1 μM) for 36 hours at theindicated concentrations. After this time, the cells were harvested andlysed, the cell lysate sonicated, centrifuged to remove any cellulardebris, and the supernatant collected. The protein concentration of eachcell lysate solution (the supernatant) was determined and the westernblot performed using standard methods.

The antibodies used were acHistone 3 (Sigma Aldrich), phospho-ATMSerine1981 (Sigma Aldrich), ATM (Sigma Aldrich), phospho-KAP-1 Serine824(Sigma Aldrich), KAP-1 (Sigma Aldrich), phosphor-p53 Serine15 (SigmaAldrich), acetyl-p53 (Sigma Aldrich), p53 (Sigma Aldrich), PARP (SigmaAldrich), cleaved-PARP (Sigma Aldrich), and β-Actin (Sigma Aldrich).

Example 1—Cell Viability

To evaluate the cytotoxicity of EDO-S101 in comparison to bendamustineand vorinostat (SAHA), HH cells were treated with either EDO-S101,bendamustine, vorinostat or an equimolar bendamustine/vorinostatcombination over a period 48 hours. Cells were treated with either 0.1μM, 1 μM, 2.5 μM, 5 μM or 10 μM solutions of the indicated compounds(FIG. 1a ).

Following treatment, cell death was evaluated by staining cells with theapoptosis markers Annexin-V and 7-AAD, and the number of apoptotic cellsquantified by flow cytometry. Annexin-V specifically targets andidentifies apoptotic cells. 7-AAD is a marker of late stage apoptotic,or necrotic cells. The number of Annexin-V and 7-AAD negative cells wascounted for each sample. Each experiment was repeated for the indicatednumber of times, and the average number of apoptosis negative cellsplotted and normalised relative to an untreated control sample (FIG. 1a). A dose response curve (FIG. 1b ) for each treatment was subsequentlyplotted, and the LD₅₀ (median lethal dose) of each treatment determined.

The equimolar combination of bendamustine and vorinotstat (LD₅₀: 1.1μM), and EDO-S101 (LD₅₀: 1.5 μM) demonstrated marked potency in HH celldeath induction after 48 hours of treatment. Both thebendamustine/vorinostat combination and EDO-S101 exhibited LD₅₀ valuesin the low micromolar range. Furthermore, both EDO-S101 and thebedamustine/vorinostat combination treatment demonstrated enhancedcytotoxicity compared to vorinostat (LD₅₀: 2.7 μM) and bendamustine(LD₅₀: 8.0 μM) as single agents.

Example 2—Western Blot Analysis of Patient T-PLL Samples

T-PLL cells (ATM mutated at L1238*, mono-allelic ATM loss, copy no=1.41)were isolated from peripheral blood (PB) of T-PLL patients were culturedin suspension, and treated with 1 μM of either bendamustine (FIG. 2,lane 2), vorinostat (lane 3), EDO-S101 (lane 5) or an equimolarcombination of vorinostat/bendamustine (lane 4) for 36 hours. After thistime, the cells were harvested, lysed and protein expression levelsdetermined by western blot analysis.

FIG. 2 shows western blots of the cell lysate for each treatment,compared to a negative control (lane 1). Staining for β-Actin was usedas a loading control for each western blot ran (rows a to k, and rows lto o respectively).

HDAC inhibitors target proteins which promote the deacetylation ofhistones, or the deacetylation of other proteins. Acetylation anddeacetylation of histones are post-translational modificationsimplicated in DNA replication and repair, and therefore the acetylationstatus of histones is crucial in cell replication pathways. Inhibitionof HDAC activity is therefore linked to induction of the DNA damageresponse. HDAC inhibitors used in these experiments include vorinostatand the fusion molecule EDO-S101.

DNA alkylating agents prevent normal DNA replication pathways fromfunctioning by binding to DNA, and therefore cause replication stress.In an attempt to repair the damage caused, the cell recruits an array ofproteins, in what is referred to as the DNA Damage response (DDR). Manyof the proteins recruited in the DDR may be used as biomarkers fordamage and replication stress. Such biomarkers include increasedexpression levels of γH2AX, phosphorylated ATM (pATM), phosphorylatedKap1 (pKap1), and stabilisation of p53. DNA alkylating agents used inthis example include bendamustine, and the fusion molecule EDO-S101(which is also a HDAC inhibitor).

Referring to FIG. 2, it is clear that treatment of T-PLL cell samplesisolated from patients with EDO-S101 led to the most significantinduction of the DDR, compared to treatment with bendamustine,vorinostat or a combination thereof. Cells treated with EDO-S101revealed the largest increase in levels of γH2AX (row l), pATM (row b),and pKAP1 (row d), all of which are heavily implicated in the DNA damageresponse. In line with the induction of the DDR, the expression levelsof Kap1 (row e) were reciprocally related to the levels of pKap1. Theseresults indicate that EDO-101 exhibits the most potent DNA alkylatingactivity compared to vorinostat and bendamustine, and furthermore, thatEDO-S101 exceeds the potency of a combination of vorinostat andbendamustine.

The induction of the DDR also causes stabilization of p53 (row h) andsubsequent phosphorylation (row f) and acetylation of p53 (acetyl-p53;row g). Referring to FIG. 2, treatment of cells with EDO-S101 resultedin the greatest accumulation of acetyl-p53 (row g) and p-p53 (row f),compared to bendamustine, vorinostat or a combination thereof. Theseresults further support EDO-S101 being the most potent inducer of DNAdamage.

Where DNA damage is extensive and the DNA cannot be repaired, p53pathways are responsible for inducing cell apoptosis. One such pathwayis characterized by the cleavage of PARP. As can be seen in FIG. 2,treatment of cells with EDO-S101 caused the greatest increase in cleavedPARP (cPARP; row j), compared to bendamustine, vorinostat, or acombination thereof. These data indicate that treatment with EDO-S101caused the most extensive and irreparable DNA damage in T-PLL cells,promoting cell apoptosis. Furthermore, these data are indicative thattreatment of T-PLL cells from patients with EDO-S101 effectivelyovercomes the protective effect conferred by stromal cells against cellapoptosis.

Referring to FIG. 2, treatment of T-PLL cells with EDO-S101 resulted inthe largest increase in acetylation of histone3 (acHistone3), comparedto vorinostat or a combination of vorinostat and bendamustine. Thesedata indicate that EDO-S101 was a more effective HDAC inhibitor thanvorinostat alone, or vorinostat in combination with bendamustine, inT-PLL cells.

In conclusion, FIG. 2 indicates that EDO-S101 induces the strongest DNAdamage response in cells. This can be attributed to its enhanced potencyas a DNA alkylator, and also as a HDAC inhibitor, compared tobendamustine or vorinostat. Furthermore, it is clear that the DNA damageand hyperacetylation induced by EDO-S101, exceeds that induced by acombination of vorinostat and bendamustine (see for example thecomparably elevated levels of pATM, acetyl-p53, pKAP1, γH2AX andacHistone 3). As a result, apoptosis in strongly induced in EDO-S101treated cells, compared to those treated with a combination ofvorinostat and bendamustine (see elevated levels of cPARP).

Example 3—Induction of Apoptosis and Resistance of EDO-S101 TreatedCells to Stromal Cell Mediated Protection in T-PLL

To further evaluate apoptosis in cells treated with EDO-S101, primaryhuman T-PLL cells were treated with either vorinostat, bendamustine,EDO-S101, or an equimolar combination of vorinostat and bendamustine, at0.01 μM, 0.1 μM, 1 μM, 5 μM or 10 μM concentrations, and incubated for48 hours. Following treatment, cell death was evaluated by stainingcells with the apoptosis markers Annexin-V and 7-AAD, and the number ofapoptotic cells quantified by flow cytometry. Each experiment wasrepeated for the indicated number of times, and the average number ofapoptosis negative cells plotted and normalised relative to an untreatedcontrol sample. A dose response curve (FIG. 3a ) for each treatment wassubsequently plotted, and the LD₅₀ (median lethal dose) of eachtreatment determined.

The LC₅₀ values for each treatment were calculated for vorinostat (20.4μM), bendamustine (7.3 μM), EDO-S101 (1.0 μM), or an equimolarcombination of vorinostat and bendamustine (4.4 μM). The LC₅₀ value forEDO-S101 was found to be lower in primary human T-PLL cells (1.0 μM)(FIG. 3a ) than in HH cells (1.5 μM) (FIG. 1b ) under comparableexperimental conditions indicating enhanced efficacy against T-PLLcells. Furthermore, EDO-S101 (1.0 μM) exhibited approximately a 4-foldincrease in potency against T-PLL cells compared to a combination ofvorinostat and bendamustine (4.4 μM).

The LC₅₀ of EDO-S101 in healthy CD3+ T-cells was determined to be 4.4μM, indicating that EDO-S101 was approximately 4-fold more potentagainst T-PLL cells compared to healthy CD3+ T-cells, under experimentalconditions. This result demonstrated that EDO-S101 had selectivity forT-PLL cells over healthy T-cells.

NKtert bone-marrow stromal cells are known to protect mutated T-cellsagainst the effects of drugs and against apoptosis. To evaluate theprotections conferred by NKtert cells to primary human T-PLL cells,NKtert cells and T-PLL cells were co-cultured for treatment withEDO-S101. Primary T-PLL cells with (FIG. 3b ) and without (FIG. 3c )co-cultures of NKtert cells were treated with increasing concentrations(0.1, 1, or 10 μM) of vorinostat, bendamustine, an equimolar combinationof vorinostat and bendamustine, or EDO-S101, and incubated for 48 hours.

Following treatment, cell death was evaluated by staining cells with theapoptosis markers Annexin-V and 7-AAD, and the number of apoptotic cellsquantified by flow cytometry. Each experiment was repeated for theindicated number of times, and the average number of apoptosis negativecells plotted as a ratio relative to an untreated control sample (FIG.3b , FIG. 3c ).

Referring to FIG. 3b , the control sample 0 on the left hand graph isnormalised to 1 for normal T-PLL cells. As can be seen from FIG. 3c thecontrol sample 0 for the T-PLL/NKtert co-cultured cells is greater than1, indicating enhanced survival of T-PLL cells in the presence of NKtertcells compared to monoculture.

Referring to FIGS. 3b and 3c , both T-PLL cells, and T-PLL/NKtertco-cultured cells, were sensitive to treatment with EDO-S101. Extensiveapoptosis was observed in both T-PLL cells, and co-cultured T-PLL/NKtertcells, when treated with 10 μM EDO-S101, with an approximate cell deathcount of greater than 95%. These data indicate that treatment of T-PLLcells with EDO-S101 overcame the protection conferred by the NKtertcells. Furthermore, T-PLL cells treated with either bendamustine,vorinostat, or an equimolar combination of bendamustine and vorinostat,were not as effective as EDO-S101 in overcoming NKtert associatedprotection co-cultured T-PLL cells. These data are further supported bythe observation that treatment of human T-PLL cells with EDO-S101 led tothe most enhanced levels of cPARP, a key indicator of cell apoptosis(FIG. 2).

It was hypothesised that EDO-S101 could be affecting the viability ofthe NKtert cells in the co-cultured T-PLL/NKtert cell experiments shownin FIG. 3c . Consequently, the effect on cell viability of NKtert bonemarrow stromal cells (BMSC) feeders cells alone was also investigated.Cells were treated with either bendamustine, vorinostat, an equimolarcombination of bendamustine and vorinostat, or EDO-S101, at either 0.1,1, 5 or 10 μM concentrations, incubated for 48 hours, and cell viabilityassessed using MTT assays (FIG. 3d ). As can be seen in FIG. 3d , thereduction in viability of NKtert cells treated with EDO-S101,vorinostat, or an equimolar combination of vorinostat and bendamustine,were largely comparable over the concentrations investigated.Bendamustine treatment did not have a pronounced concentration dependenteffect on the viability of NKtert cells. Importantly, the viability ofcells treated with EDO-S101 and vorinostat at 10 μM were comparable,providing confidence that the cell death induced by treatment ofT-PLL/NKtert co-cultured cells with EDO-S101 (FIG. 3c ) was not a resultof reduced viability of NKtert cells (FIG. 3d ).

Example 4—In Vivo Analysis of Leukemic Blood Leukocytes (WBC) CountFollowing Treatment with EDO-S101

Mice were injected with leukaemia cells derived from CD2-MTCP1 mice, aspreviously described (FIG. 4a ).

CD2-MTCP1 cells are an aggressive, transplantable subline and aresuitable for in vivo analysis as a T-PLL like model. At day 10post-transplantation of CD2-MTCP1 cells by intraperitoneal injection,mice with comparable leukemic blood leukocytes (WBC) counts were dividedinto four groups at random. Each group was intravenously administeredeither fludarabine (34 mg/kg), bendamustine (Day 10, 60 mg/kg; Day15-21, 20 mg/kg) or EDO-S101 (Day 10, 50 mg/kg; Day 15-21, 20 mg/kg) atthe indicated doses on Day 10, Day 15, Day 17, Day 19 and Day 21post-transplantation. Samples of blood were taken at regular intervals(Day 9 and Day 14 post-transplantation), and the mice sacrificed at 22days post-transplantation (FIG. 4a ).

Fludarabine was selected for experiments as a comparative compound, andis a FDA approved chemotherapy for the treatment of leukemia andlymphoma. Fludarabine is a purine derivative, and interferes with thereplication of DNA. It is on the World Health Organisation's List ofEssential Medicines.

The blood samples taken were analysed for leukemic blood leukocytelevels (WBC), and the average cell count in each group determined.Comparison of WBC count at Day 14 and Day 9, revealed that bendamustineand EDO-S101 significantly delayed the increase in WBC cells compared toa control sample and fludarabine (FIG. 4b ). These data indicated thatEDO-S101 and bendamustine was delaying the onset of disease progressionin the recipient mice.

Following sacrifice of the mice on day 22 post-transplantation, thepost-mortem spleen weights of each mouse were determined (FIG. 4c ). Thereduced average spleen weight in mouse cohorts treated with bendamustineor EDO-S101, compared to cohorts treated with fludarabine or a control,corroborate the findings previously discussed in FIG. 4b . Tumourmanifestation was therefore shown to be less advanced in cohorts treatedwith EDO-S101 or single-agent bendamustine, compared to fludarabinetreated or control cohorts.

Example 5—In Vivo Analysis of Leukemic Blood Leukocytes (WBC) CountFollowing Treatment with EDO-S101

Mice were injected with leukaemia/lymphoma cells derived from ΔJAK1mice, as previously described (FIG. 5). ΔJAK1 is a model for matureT-cell lymphoma. Mice were divided into four groups at randompost-transplantation. Each group was intravenously administered eitherfludarabine (18 mg/kg), bendamustine (18 mg/kg) or EDO-S101 (18 mg/kg)at the indicated doses on Day 7, Day 10, Day 13, Day 17 and Day 22. Thepercentage survival of each cohort was plotted as a function of time(FIG. 5). The overall survival (mean survival 26 days) of mice treatedwith EDO-S101 was found to be significantly prolonged compared tocontrol mice (mean survival 19 days), bendamustine (mean survival 18days) or fludarabine (mean survival 19 days). These data indicate thattreatment with EDO-S101 has a positive effect on the overall survival ofmice with T-cell lymphoma, increasing the average survival time by asmuch as 8 days compared to bendamustine or fludarabine.

The invention claimed is:
 1. A method of treating T-cell prolymphocyticleukemia (T-PLL) in a patient in need thereof, comprising administeringto said patient an effective amount of tinostamustine or apharmaceutically acceptable salt thereof.
 2. The method according toclaim 1, wherein the T-PLL is relapsed, refractory T-PLL, or acombination thereof.
 3. The method according to claim 1, wherein theT-PLL is metastatic.
 4. The method according to claim 1, wherein theT-PLL is advanced.
 5. The method according to claim 1, whereintinostamustine or a pharmaceutically acceptable salt thereof isadministered intravenously to the patient in need thereof at a dosagelevel of from 0.3 mg/m² to 300 mg/m² body surface area of the patient.6. The method according to claim 1, wherein tinostamustine or apharmaceutically acceptable salt thereof is administered intravenouslyto the patient in need thereof on day 1 of a treatment cycle.
 7. Themethod according to claim 1, wherein tinostamustine or apharmaceutically acceptable salt thereof is administered intravenouslyto the patient in need thereof over an infusion time of 60 minutes. 8.The method according to claim 1, wherein tinostamustine or apharmaceutically acceptable salt thereof is administered intravenouslyto the patient in need thereof at a dosage level of from 20 mg/m² to 150mg/m² body surface area of the patient, on day 1 of a 21 day treatmentcycle, over an infusion time of 60 minutes.
 9. The method according toclaim 1, wherein the patient is treated with tinostamustine or apharmaceutically acceptable salt thereof and radiotherapy.
 10. Themethod according to claim 9, wherein said radiotherapy treatment isgiven to the patient in need thereof at a dose of 1 to 5 Gy over 5-10consecutive days.
 11. The method according to claim 5, whereintinostamustine or a pharmaceutically acceptable salt thereof isadministered at a dosage level of from 20 mg/m² to 150 mg/m² bodysurface area of the patient.
 12. The method according to claim 6,wherein tinostamustine or a pharmaceutically acceptable salt thereof isadministered on day 1 of a 21 day treatment cycle.
 13. The methodaccording to claim 1, wherein tinostamustine or a pharmaceuticallyacceptable salt thereof is administered intravenously to the patient inneed thereof over an infusion time of 45 minutes.
 14. The methodaccording to claim 1, wherein tinostamustine or a pharmaceuticallyacceptable salt thereof is administered intravenously to the patient inneed thereof over an infusion time of 30 minutes.
 15. The methodaccording to claim 10, wherein said radiotherapy treatment is given tothe patient at a dose of 2 Gy over 5-10 consecutive days.